CN110964790B - Method for detecting tea-goat PIGY gene CNV marker and application thereof - Google Patents
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Abstract
The invention discloses a method for detecting a tea-card sheep PIGY gene CNV marker and application thereof: based on a real-time fluorescence quantitative PCR technology, the genomic DNA of the tea-goat is used as a template, a pair of specific primers is used for amplifying partial fragments of the copy number variation region of the PIGY gene of the tea-goat, the other pair of specific primers is used for amplifying partial fragments of the ANKRD1 gene of the tea-goat as a reference, and then 2*2 ‑ΔCt The method calculates the copy number variation type of an individual, and according to the correlation analysis result of the copy number variation and the growth traits of the tea-goat, the method provided by the invention lays a foundation for establishing the dominant population of the growth traits by using the PIGY gene CNV marker of the tea-goat, and is favorable for accelerating the molecular marker-assisted selective breeding process.
Description
Technical Field
The invention belongs to the field of molecular genetic breeding, and particularly relates to a method for detecting PIGY gene copy number variation of a tea-goat, which utilizes a real-time quantitative PCR technology, takes genomic DNA of the tea-goat as a template and takes a single-copy ANKRD1 gene as a reference according to 2*2 -ΔCt The values determine the type of variation of the PIGY gene copy number of the individual.
Background
With the intensive genome research, there is evidence that Copy Number Variations (CNVs) affect gene networks and regulate expression of related genes, contributing to variability of individual phenotypes, and thus the use of candidate markers for CNVs associated with size traits can accelerate sheep genetic breeding progress. Copy number variation generally refers to structural variation of more than 50bp across the entire genome, resulting from rearrangements occurring within the genome.
At present, there are three major methods for detecting CNVs in the whole genome range of human and animals, which are microarray-based comparative genomic hybridization (aCGH), next-Generation Sequencing (NGS), and SNP chip technologies. Oligonucleotide probe chips are widely used in comparative genome hybridization chips, and have the characteristics of high sensitivity, high precision and small sample size; the bacterial artificial chromosome chip has high manufacturing cost, low resolution and long time consumption, and is rarely used. In addition, existing chip platforms are inefficient at detecting new copy number variations. With the development of the next generation sequencing technology, the most effective detection means is to detect the genome structure variation by re-sequencing, but the method has higher cost compared with other methods.
Currently, there are two main methods for detecting CNVs known in the genome, namely PCR-based detection techniques and hybridization-based detection techniques. The PCR detection technology mainly comprises Real-time fluorescent Quantitative PCR (Real-time qPCR), ligation-dependent Multiplex amplification probe hybridization (MLPA) and short-fragment Multiplex Quantitative PCR (QMPSF). At present, the most widely used method is qPCR technology, and the method has the advantages of high sensitivity, simple operation method, high speed, good repeatability and less pollution. Hybridization techniques mainly include Southern blotting hybridization, fluorescence In Situ Hybridization (FISH), multiplex Amplification Probe Hybridization (MAPH), etc., but these methods have high cost, long detection time and are not accurate enough, and are used less at present.
The tea-goat is a good sheep variety produced from Wulan county of Tibetan autonomous state of Mongolia of Hai-West Mongolia of Qinghai province through long-term natural selection and artificial breeding. The further breeding improvement of the tea-card sheep has an important effect on the industrialization and economic benefits of the tea-card sheep. The PIGY (phosphatidylinositol) gene is one of the members of the PIG gene family, is located on sheep chromosome 6, and the expression product of the PIGY gene is combined with the expression proteins of the PIGA, PIGC, PIGH, PIGP, PIGQ and DPM2 genes to form a GPI-GnT complex which initiates the biosynthesis of Glycosylphosphatidylinositol (GPI). GPI is synthesized on the endoplasmic reticulum and is used for anchoring many surface proteins. Proteins containing GPI anchors play an important role in cell-cell interactions. At present, only the PIGY gene is found to overlap with a plurality of quantitative trait sites related to muscle density, carcass weight and the like, and the analysis and application of the sheep PIGY gene CNV are still in a blank stage.
Disclosure of Invention
The invention aims to provide a method for detecting a tea-card sheep PIGY gene CNV marker and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting a tea-goat PIGY gene CNV marker comprises the following steps:
taking the whole genome DNA of the individual Pigy of the tea-goat as a template, taking a primer pair PIGY-CNV and a primer pair ANKRD1-CNV as primers, respectively amplifying a copy number variation region of the PIGY gene and a partial fragment of the ANKRD1 gene serving as an internal reference through real-time fluorescence quantitative PCR, and then identifying the copy number variation type of the individual PIGY gene of the tea-goat according to a quantitative result;
the PIGY-CNV primer pair comprises:
an upstream primer F1:5'-AGAGTGGCGGGTGATAAGTG-3'
A downstream primer R1:5'-CAGTCCTGCCAAAGACACCA-3';
the primer pair ANKRD1-CNV is as follows:
an upstream primer F2:5'-TGGGCACCACGAAATTCTCA-3'
A downstream primer R2:5'-TGGCAGAAATGTGCGAACG-3'.
Preferably, the copy number variation region of the PIGY gene is located in sheep reference genome sequence NC-019463.236121601 bp-36125200bp.
Preferably, the type of copy number variation is based on2*2 -ΔCt The quantitative results were divided into three categories: insertion type, 2*2 -ΔCt >2.5; deletion form, 2*2 -ΔCt <1.5; normal, 2*2 -ΔCt ＝1.5～2.5。
Preferably, the amplification system for real-time fluorescent quantitative PCR comprises: 50 ng/. Mu.L template DNA 1. Mu.L, 10pmol/L primer pair PIGY-CNV or primer pair ANKRD1-CNV corresponding upstream and downstream primers 0.5. Mu.L each, 2 XSSYBR Green qPCR Mix 5. Mu.L and ddH 2 O 3μL。
Preferably, the reaction procedure for the real-time fluorescent quantitative PCR comprises the following steps: pre-denaturation at 95 ℃ for 1min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, plate reading, a total of 39 cycles.
Preferably, the size of the fragment amplified based on the PIGY-CNV primer pair is 90bp, and the size of the fragment amplified based on the ANKRD1-CNV primer pair is 143bp.
The method for detecting the tea-goat PIGY gene CNV marker is applied to the tea-goat molecular marker-assisted selective breeding.
Preferably, in the tea-card sheep, the individuals with the insertion type copy number variation types are superior to the individuals with the deletion type and normal type copy number variation types in growth traits such as weight, chest circumference and the like.
The invention has the beneficial effects that:
the method for detecting the PIGY gene CNV marker provided by the invention can be used for quickly establishing the dominant population of the growth traits (such as weight and chest circumference) of the tea-cartoon sheep according to the detection result and the correlation analysis result of the copy number variation condition in the tea-cartoon sheep group and the important economic traits such as weight, chest circumference, height, length and the like by using the genomic DNA of the tea-cartoon sheep to be detected as a template and utilizing real-time fluorescence quantitative PCR to detect the copy number variation type of the PIGY gene in the genome. The method is simple and rapid, and is convenient for popularization and application.
Compared with the prior art, the invention has the following advantages:
(1) The detection method for the tea-Cal sheep PIGY gene CNV marker is not limited by age, can be used for early breeding of tea-Cal sheep individuals, and can be selected even just after birth;
(2) The method can accurately and reliably detect the PIGY gene copy number variation of the tea-goat, and is simple and convenient to operate;
(3) The detection of the tea-card sheep PIGY gene CNV marker provides scientific basis for the molecular marker-assisted selection of the growth traits of the tea-card sheep PIGY gene CNV marker.
Drawings
FIG. 1 is a distribution diagram of copy number variation of the PIGY gene of 312 Cauchard sheep.
FIG. 2 is a graph plotting amplification curves for qPCR (PIGY gene) performed in the present invention.
FIG. 3 is a dissolution profile plotted for qPCR (PIGY gene) performed in the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
The invention utilizes real-time fluorescence quantitative PCR to detect copy number variation of PIGY gene of tea-goat and apply the variation to molecular breeding, and comprises the following steps:
(1) Designing a primer according to a sheep PIGY gene sequence of an NCBI database, and detecting the primer by using a common PCR;
(2) Detecting the copy number variation condition of the candidate sites in the population by adopting a real-time fluorescent quantitative PCR (qPCR) technology;
(3) Performing correlation analysis on the copy number variation type and the growth traits of the tea-goat by using SPSS 23.0 software, and screening a CNV (CNV) marker related to the growth traits of the tea-goat;
(4) And (4) breeding the tea-card sheep with excellent growth characters according to the copy number variation type.
The specific process for detecting the copy number variation of the PIGY gene of the tea-goat by utilizing real-time fluorescent quantitative PCR comprises the following steps:
1. sheep sample collection
The method specifically uses 312 tea goats as detection objects, and the tea goat blood samples are collected from a tea cat town farm in Wulan county, haizhou, qinghai province (collected in 5 months in 2018).
2. Extraction of genomic DNA from blood samples
(1) Thawing frozen blood sample (mainly blood cells) at room temperature, sucking 500 μ L of blood into a 1.5mL centrifuge tube, adding Phosphate Buffer Solution (PBS) with equal volume, mixing, gently shaking, centrifuging at 4 deg.C and 12000r/min for 5min, and discarding supernatant; this procedure was repeated until the supernatant was clear.
(2) Adding 500 mu L of DNA extraction buffer solution into a centrifuge tube, gently blowing and beating to separate the blood cell sediment from the wall of the centrifuge tube, and carrying out water bath at 37 ℃ for 1h.
(3) Adding proteinase K to 5 μ L (20 mg/mL), mixing, digesting overnight (about 16 h) in 55 deg.C water bath until no flocculent precipitate is observed, clarifying the solution, adding 10 μ L proteinase K, mixing, and digesting until it is clear.
(4) Cooling the reaction solution to room temperature, adding 500 mu L Tris saturated phenol, gently shaking for 15min to fully mix the Tris saturated phenol and the Tris saturated phenol, centrifuging the mixture at 4 ℃ at 12000r/min for 10min, and transferring the upper-layer water phase into another sterilized centrifuge tube; repeat step 1 time.
(5) Adding 500mL of chloroform, gently shaking for 20min to mix well, centrifuging at 4 deg.C and 12000r/min for 15min, and transferring the upper aqueous phase into another sterilized 1.5mL centrifuge tube.
(6) 500mL of a chloroform/isoamyl alcohol mixture (24).
(7) Add 0.1 volume NaAc buffer and 2 volumes of ice-cold absolute ethanol, mix and rotate the centrifuge tube until white flocculent precipitate separates out.
(8) Centrifugation was carried out at 12000r/min at 4 ℃ for 10min, the supernatant was discarded, and the DNA precipitate was rinsed 2 times with 70% ice-cold ethanol.
(9) Centrifuging at 4 deg.C and 12000r/min for 10min, removing supernatant, and volatilizing ethanol at room temperature.
(10) Adding 80-100 mu L of TE into the dried DNA, storing at 4 ℃ until the DNA is completely dissolved, detecting the concentration and the quality by using an ultraviolet spectrophotometer and electrophoresis, and storing at-80 ℃.
3. Amplification of target and reference sequences
A sheep PIGY gene sequence (GenBank accession No. NC-019463.2) published by NCBI database (http:// www.ncbi.nlm.nih.gov /) is taken as a reference sequence (Chr 6. The internal reference sequence is a known sequence without copy number variation, namely a 143bp sequence in the ANKRD1 gene, and the sequence information of the primer pair is shown in Table 1 (design completion time 2019, 8 months).
TABLE 1 primer information for real-time fluorescent quantitative PCR
The amplification system for real-time fluorescent quantitative PCR is calculated by 10.0 μ L as: 50 ng/. Mu.L template DNA (genomic DNA extracted from blood sample) 1. Mu.L, 10pmol/L upstream primer 0.5. Mu.L, 10pmol/L downstream primer 0.5. Mu.L, 2 XSSYBR Green qPCR Mix 5.0. Mu.L, and ddH 2 O 3.0μL。
The reaction procedure for qPCR amplification was: pre-denaturation at 95 ℃ for 1min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, plate reading (Plate Read), for a total of 39 cycles; the dissolution profile was started at 65 ℃ with 0.5 ℃ increase per cycle, raised to 95 ℃ for 5s, and the plate was read.
Primers were determined to be suitable for qPCR analysis by plotting amplification curves (figure 2) and melting peaks. According to the drawn melting curve, the curves of the samples are matched together, and the curves are smooth in trend, high and sharp in peak height, and free from primer dimer or a hybrid peak caused by nonspecific amplification (figure 3).
4. Determination of copy number variation
Each sample was amplified with primers for the target sequence and the internal reference sequence, respectively, and 3 replicates were performed for each pair of primers. According to 2*2 -ΔCt The method performs analysis of copy number variation. Wherein Δ Ct = Ct Target gene –Ct Reference gene 。2*2 -ΔCt The number of copies is indicated. C T Namely Cycle threshold, amplified by PCRThe number of amplification cycles that pass when the fluorescence signal of the amplification product reaches a set threshold.
According to 2*2 -ΔCt The quantitative results were classified into three categories: insertion type, 2*2 -ΔCt >2.5; deletion type, 2*2 -ΔCt <1.5; normal, 2*2 -ΔCt = 1.5-2.5, see fig. 1.
5. Correlation analysis of PIGY gene CNV locus and growth traits
Production data: height, length, chest circumference, and weight.
And (3) correlation analysis model: firstly, performing description analysis on data to determine whether an outlier exists, and then correcting the data by using least square analysis; according to the data characteristics, the SPSS 23.0 software was used to analyze the effect of production traits among genotypes. A fixed model was used in the analysis of genotype effects:
Y ijk ＝μ+A i +CNV j +e ijk
wherein: y is ijk For trait observations, μ is the overall mean, A i Is age of the ith individual, CNV j As a fixed effect of the jth copy number variation type, e ijk Is a random error. The variability between each set of data was examined using multiple comparisons of LSDs and the results were expressed as Mean ± SE.
Correlation analysis results show (see table 2): the individuals with the CNV type of insertion on the PIGY gene of the tea-goat are obviously superior to the normal individuals and the deletion individuals in growth traits (weight and chest circumference); the results show that the insertion type of the PIGY gene CNV locus can be used as a candidate molecular genetic marker (CNV marker) for improving the growth traits of the tea-goat, and is used for early molecular breeding of the tea-goat.
TABLE 2 analysis of the relationship between copy number variation of the PIGY gene of Cauchard sheep and growth traits
Note: mean values are marked with the same letter indicating that the difference is not significant (P)>0.05 Average) ofThe difference in the letters on the shoulder of the value indicates a significant difference (abc: P)<0.05；ABC:P<0.01)。 * P<0.05； ** P<0.01。
6. Application of CNV marker in tea-card sheep breeding
The obtained CNV candidate molecular genetic marker can be used for searching quantitative trait loci which are related to the CNV candidate molecular genetic marker or are closely linked with the CNV candidate molecular genetic marker and influence the growth traits of the tea-goat. The method can also be used for molecular marker-assisted selection of the tea-Calf sheep, namely, the inserted individual tea-Calf sheep is selected for reservation and propagation by detecting the copy number variation type of the CNV locus of the PIGY gene of the tea-Calf sheep, so that the breeding process of the improvement of the variety of the tea-Calf sheep can be accelerated.
<110> northwest agriculture and forestry science and technology university
<120> method for detecting tea-card sheep PIGY gene CNV marker and application thereof
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Claims (6)
1. Detect tea card sheepPIGYA method for marking gene CNV, which is characterized in that: the method comprises the following steps:
taking the genomic DNA of the goat with the tea card as a template, taking the primer pair PIGY-CNV and the primer pair ANKRD1-CNV as primers, and respectively amplifying by real-time fluorescence quantitative PCRPIGYCopy number variation region of gene and gene serving as internal referenceANKRD1Partial segments of the gene, and then identifying the tea goat according to the quantitative resultPIGYCopy number variation type of gene;
the PIGY-CNV primer pair comprises:
an upstream primer F1:5'-AGAGTGGCGGGTGATAAGTG-3'
A downstream primer R1:5'-CAGTCCTGCCAAAGACACCA-3';
the primer pair ANKRD1-CNV is as follows:
an upstream primer F2:5'-TGGGCACCACGAAATTCTCA-3'
A downstream primer R2: 5'-TGGCAGAAATGTGCGAACG-3';
saidPIGYThe copy number variation region of the gene is positioned in a sheep reference genome sequence NC-019463.2 36121601bp-36125200 bp;
the copy number variation type is according to 2*2 -ΔCt The quantitative results were divided into three categories: insertion type, 2*2 -ΔCt >2.5; deletion form, 2*2 -ΔCt <1.5; normal, 2*2 -ΔCt =1.5~2.5。
2. The method of claim 1, wherein the tea-goat is a goat for detecting the presence of the protein in the bloodPIGYA method for marking gene CNV, which is characterized in that: the real-time fluorescent quantitative PCR amplification system comprises 1 mu L of template DNA of 50 ng/mu L and 0.5 mu L of upstream and downstream primers corresponding to a primer pair PIGY-CNV or a primer pair ANKRD1-CNV of 10pmol/L respectively.
3. The method of claim 1, wherein the tea-goat is a goat for detecting the presence of the protein in the bloodPIGYA method for marking gene CNV, which is characterized in that: the reaction program of the real-time fluorescence quantitative PCR comprises the following steps: 95. pre-denaturation at deg.C for 1min; 95. the denaturation at 15s and 60 deg.CAnnealing for 15s, extension at 72 ℃ for 30s, for 39 cycles.
4. The method of claim 1, wherein the tea-goat is a goat for detecting the presence of the protein in the bloodPIGYMethod for marking a gene CNV, characterized in that: the amplified fragment size based on the primer pair PIGY-CNV was 90bp, and the amplified fragment size based on the primer pair ANKRD1-CNV was 143bp.
5. The use of the method according to any one of claims 1 to 4 in molecular marker assisted selection breeding of theacrine characterized in that: the growth traits of individuals with insertion type in the tea-card sheep are superior to those of individuals with deletion type and normal type, and the growth traits are weight and/or chest circumference.
6. Detect tea card sheepPIGYThe real-time fluorescent quantitative PCR kit marked by the gene CNV is characterized in that: the kit comprises a primer pair PIGY-CNV and a primer pair ANKRD1-CNV;
the PIGY-CNV primer pair comprises:
an upstream primer F1:5'-AGAGTGGCGGGTGATAAGTG-3'
A downstream primer R1:5'-CAGTCCTGCCAAAGACACCA-3';
the primer pair ANKRD1-CNV is as follows:
an upstream primer F2:5'-TGGGCACCACGAAATTCTCA-3'
A downstream primer R2:5'-TGGCAGAAATGTGCGAACG-3'.
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